Liver disease represents a major global medical problem. Understanding the genetic basis for liver cell-fate determination will provide insight into diagnosis and treatment/prevention of liver disease. The goal of this proposal is to investigate the genetic basis for mammalian liver specification using combined molecular and somatic cell genetic approaches. A liver-specific hierarchical regulatory cascade has recently been identified in which transcription factor HNF4 drives expression transcription factor HNF1 which is responsible for activating expression of a number of essential liver genes. The investigators have recently isolated and characterized hepatoma "variant" cell lines containing specific and distinct regulatory defects in this HNF4:HNF1 hierarchical pathway. One variant line, lacks HNF4 mRNA and fail to express HNF1 or HNF1-responsive genes, though expression of other liver genes is unaffected. Ectopic expression of cloned HNF4 in H11 cells rescues expression of endogenous HNF1 and HNF1-responsive genes. Other variant lines have more profound defects which cannot be rescued cloned transacting factors. Hepatoma variant line H11, harboring apparently simple regulatory defect(s), will be used to analyze three aspects of the HNF4:HNF1 cascade. First, the in vivo contribution of HNF1 and/or HNF4 in driving expression of a panel of endogenous liver, specific genes will be assessed using H11 cells stably expressing cloned HNF1 and/or HNF4. Secondly, the influence of HNF1 and HNF4 on the state of chromatin at the alpha1AT locus will be examined using DNase I hypersensitivity assays. Thirdly, genetic loci capable of transactivating the HNF4:HNF1 pathway will be identified using microcell-mediated chromosome transfer. Finally, characterization of additional variants is being carried out to establish complementation groups in order to identify the components of regulatory circuitry in the hepatocyte. Determining the role of the HNF4:HNF1 cascade on the liver phenotype as well as identifying the factors which drive this pathway will yield essential information required to understand how liver gene expression is activated and maintained in the hepatocyte.